scholarly journals Light and electron microscopic localization of D-aspartate oxidase in peroxisomes of bovine kidney and liver: an immunocytochemical study.

1996 ◽  
Vol 44 (9) ◽  
pp. 1013-1019 ◽  
Author(s):  
K Zaar
Keyword(s):  
Protein A ◽  
Cell Types ◽  
Subcellular Fractionation ◽  
Systematic Investigation ◽  
Kidney Cortex ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Peroxisomal Enzyme ◽  
Bovine Kidney

D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.

scholarly journals Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes.

1986 ◽  
Vol 34 (12) ◽  
pp. 1709-1718 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Proximal Tubule ◽  
Rat Kidney ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Thin Sections ◽  
Gold Particles

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.

scholarly journals d-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes

1989 ◽  
Vol 261 (1) ◽  
pp. 233-238 ◽  
Author(s):  
K Zaar ◽  
A Völkl ◽  
H D Fahimi
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Matrix Protein ◽  
Subcellular Fractionation ◽  
Matrix Proteins ◽  
Kidney Cortex ◽  
Acid Oxidase ◽  
Marker Enzymes ◽  
Freezing And Thawing ◽  
Amino Acid Oxidase

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.

scholarly journals D-aspartate oxidation by rat and bovine renal peroxisomes: an electron microscopic cytochemical study.

1990 ◽  
Vol 38 (9) ◽  
pp. 1377-1381 ◽  
Author(s):  
M E Beard
Keyword(s):  
Amino Acid ◽  
Oxidase Activity ◽  
Electron Microscopic Level ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
Cytochemical Localization ◽  
Peroxisomal Enzyme ◽  
Cytochemical Study ◽  
Biochemical Studies

D-amino acid oxidase, a peroxisomal enzyme, and D-aspartate oxidase, a potential peroxisomal enzyme, share biochemical attributes. Both produce hydrogen peroxide in flavin-requiring oxidative reactions. Such similarities suggest that D-aspartate oxidase may also be localized to peroxisomes. Definitive identification of D-aspartate oxidase as a peroxisomal enzyme depends, however, on visualization at the electron microscopic level. Using incubation conditions shown to be specific for the enzyme in biochemical studies, this report extends the cytochemical localization of D-amino acid oxidase to bovine renal peroxisomes, and shows that D-aspartate can be oxidized by rat and bovine renal peroxisomes. An unexpected finding was the sensitivity of both D-amino acid oxidase activity (proline specific) and D-aspartate oxidase activity to inhibition by agents used in biochemical studies to discriminate between the two enzyme activities. Therefore, it is possible that, in the cytochemical system used in this study, (a) either D-proline and D-aspartate are substrates for only one enzyme or (b) the two enzymes have additional overlapping biochemical properties.

scholarly journals cDNA cloning and expression of the flavoprotein d-aspartate oxidase from bovine kidney cortex

1997 ◽  
Vol 322 (3) ◽  
pp. 729-735 ◽  
Author(s):  
Tatjana SIMONIC ◽  
Stefano DUGA ◽  
Armando NEGRI ◽  
Gabriella TEDESCHI ◽  
Massimo MALCOVATI ◽  
...  
Keyword(s):  
Active Enzyme ◽  
Cloning And Expression ◽  
Kidney Cortex ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Peroxisomal Enzyme ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
First Time ◽  
Peroxisomal Targeting Signal

The isolation and sequencing of the complete cDNA coding for a d-aspartate oxidase, as well as the overexpression of the recombinant active enzyme, are reported for the first time. This 2022 bp cDNA, beside the coding portion, comprises a 5´ untranslated tract and the whole 3´ region including the polyadenylation signal and the poly(A) tail. The encoded protein comprises 341 amino acids, with the last three residues (-Ser-Lys-Leu) representing a peroxisomal targeting signal 1 (PTS1), hitherto unknown for this protein. The overexpression of recombinant d-aspartate oxidase was achieved in a prokaryotic system, and a soluble and active enzyme was obtained which accounted for about 10% of total bacterial protein. Comparisons with the known cDNAs for mammalian d-amino acid oxidase, another peroxisomal enzyme, are also made. The close structural and functional similarities shared by these enzymes at the protein level are not reflected at the nucleic acid level.

scholarly journals Structure, composition, physical properties, and turnover of proliferated peroxisomes. A study of the trophic effects of Su-13437 on rat liver.

1975 ◽  
Vol 67 (2) ◽  
pp. 281-309 ◽  
Author(s):  
F Leighton ◽  
L Coloma ◽  
C Koenig
Keyword(s):  
Enzyme Activities ◽  
Average Diameter ◽  
Urate Oxidase ◽  
Leucine Incorporation ◽  
Acid Oxidase ◽  
Peroxisome Proliferation ◽  
Amino Acid Oxidase ◽  
Liver Size ◽  
Peroxisomal Enzyme ◽  
Trophic Effect

Peroxisome proliferation has been induced with 2-methyl-2-(p-[1,2,3,4-tetrahydro-1-naphthyl]-phenoxy)-propionic acid (Su-13437). DNA, protein, cytochrome oxidase, glucose-6-phosphatase, and acid phosphatase concentrations remain almost constant. Peroxisomal enzyme activities change to approximately 165%, 50%, 30%, and 0% of the controls for catalase, urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase, respectively. For catalase the change results from a decrease in particle-bound activity and a fivefold increase in soluble activity. The average diameter of peroxisome sections is 0.58 +/- 0.15 mum in controls and 0.73 +/- 0.25 mum after treatment. Therefore, the measured peroxisomal enzymes are highly diluted in proliferated particles. After tissue fractionation, approximately one-half of the normal peroxisomes and all proliferated peroxisomes show matric extraction with ghost formation, but no change in size. In homogenates submitted to mechanical stress, proliferated peroxisomes do not reveal increased fragility; unexpectedly, Su-13437 stabilizes lysosomes. Our results suggest that matrix extraction and increased soluble enzyme activities result from transmembrane passage of peroxisomal proteins. The changes in concentration of peroxisomal oxidases and soluble catalase after Su-13437 allow the calculation of their half-lives. These are the same as those found for total catalase, in normal and treated rats, after allyl isopropyl acetamide: about 1.3 days, a result compatible with peroxisome degradation by autophagy. A sequential increase in liver RNA concentration, [14C]leucine incorporation into DOC-soluble proteins and into immunoprecipitable catalase, and an increase in liver size and peroxisomal volume per gram liver, characterize the trophic effect of the drug used. In males, Su-13437 is more active than CPIB, another peroxisome proliferation-inducing drug; in females, only Su-13437 is active.

scholarly journals Immunoelectron microscopic study of a new D-amino acid oxidase-immunoreactive subcompartment in rat liver peroxisomes.

1991 ◽  
Vol 39 (1) ◽  
pp. 95-102 ◽  
Author(s):  
N Usuda ◽  
S Yokota ◽  
R Ichikawa ◽  
T Hashimoto ◽  
T Nagata
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Small Area ◽  
Electron Microscopic Observation ◽  
Glycolate Oxidase ◽  
Acid Oxidase ◽  
Electron Microscopic ◽  
Amino Acid Oxidase ◽  
The Matrix ◽  
Electron Lucent

We report the presence of a new subcompartment in rat liver peroxisomal matrix in which only D-amino acid oxidase is localized and other matrix enzymes are absent. By electron microscopic observation, the rat liver peroxisome has generally been considered to consist of a single limiting membrane, an electron-dense crystalline core, and a homogeneous matrix. Immunohistochemical staining for D-amino acid oxidase by the protein A-gold technique revealed the presence of a small area in the matrix that was immunoreactive for the enzyme and was less electron-dense than the surrounding matrix. The localization of D-amino acid oxidase in this small area of the peroxisomal matrix was confirmed by immunoelectron microscopy on freeze-substituted tissues processed without chemical fixation. To analyze the characteristics of the electron-lucent area, immunoreactivity for various peroxisomal enzymes, including catalase, acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, 3-ketoacyl-CoA thiolase, L-alpha-hydroxy acid oxidase (isozyme B), and glycolate oxidase (isozyme A), was assayed. The electron-lucent area was negative for all of these. By double staining for D-amino acid oxidase and catalase, using colloidal gold particles of different sizes, these enzymes were shown to be located in separate areas in the matrix.

scholarly journals Immunochemical characterization of l-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues

1995 ◽  
Vol 309 (3) ◽  
pp. 993-998 ◽  
Author(s):  
D Boivin ◽  
D Bilodeau ◽  
R Béliveau
Keyword(s):  
Polyclonal Antibodies ◽  
Protein A ◽  
Cell Types ◽  
Critical Micellar Concentration ◽  
Kidney Cortex ◽  
Rat Tissues ◽  
Carboxyl Methyltransferase ◽  
C Terminus ◽  
Protein Carboxyl Methyltransferase ◽  
Mammalian Tissues

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.

scholarly journals The N –methyl D–aspartate receptor glycine site and D–serine metabolism: an evolutionary perspective

2004 ◽  
Vol 359 (1446) ◽  
pp. 943-964 ◽  
Author(s):  
Michael J. Schell
Keyword(s):  
Nmda Receptor ◽  
Divergence Time ◽  
Postnatal Period ◽  
Serine Racemase ◽  
Acid Oxidase ◽  
Glycine Site ◽  
Amino Acid Oxidase ◽  
Sequence Alignments ◽  
Peroxisomal Enzyme ◽  
Serine Metabolism

The N –methyl D–aspartate (NMDA) type of glutamate receptor requires two distinct agonists to operate. Glycine is assumed to be the endogenous ligand for the NMDA receptor glycine site, but this notion has been challenged by the discovery of high levels of endogenous D–serine in the mammalian forebrain. I have outlined an evolutionary framework for the appearance of a glycine site in animals and the metabolic events leading to high levels of D–serine in brain. Sequence alignments of the glycine–binding regions, along with the scant experimental data available, suggest that the properties of invertebrate NMDA receptor glycine sites are probably different from those in vertebrates. The synthesis of D–serine in brain is due to a pyridoxal–5'–phosphate (B 6 )–requiring serine racemase in glia. Although it remains unknown when serine racemase first evolved, data concerning the evolution of B 6 enzymes, along with the known occurrences of serine racemases in animals, point to D–serine synthesis arising around the divergence time of arthropods. D–Serine catabolism occurs via the ancient peroxisomal enzyme D–amino acid oxidase (DAO), whose ontogenetic expression in the hindbrain of mammals is delayed until the postnatal period and absent from the forebrain. The phylogeny of D–serine metabolism has relevance to our understanding of brain ontogeny, schizophrenia and neurotransmitter dynamics.

scholarly journals Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.

1979 ◽  
Vol 27 (3) ◽  
pp. 735-745 ◽  
Author(s):  
G Arnold ◽  
L Liscum ◽  
E Holtzman
Keyword(s):  
Nervous System ◽  
Amino Acid ◽  
Kojic Acid ◽  
Ultrastructural Localization ◽  
Cell Types ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Marked Diminution ◽  
Biochemical Studies ◽  
System Cell

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.

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